Compositions and methods for promoting nerve regeneration

ABSTRACT

FK506 and geldanamycin promote nerve regeneration by a common mechanism that involves the binding of these compounds to polypeptide components of steroid receptor complexes other than the steroid hormone binding portion of the complex (FKBPS52 and hsp90, respectively). These and other agents cause hsp90 dissociation from steroid receptor complexes or block association of hsp90 with steroid receptor complexes.

BACKGROUND OF THE INVENTION

[0001] Following traumatic or mechanically induced axonal degenerationin the peripheral nervous system, axonal regeneration ensues, resultingin functional recovery. However, the rate of axonal elongation (3-4mm/day) is slow. Consequently, recovery is measured in weeks or months,depending upon the distance between the site of injury and the targettissue. Therapies that speed regeneration over long distances would behighly beneficial to patients and would significantly reduce health carecosts.

[0002] The immunosuppressant drug FK506 (USAN tacrolimus; Prograf®)speeds functional recovery and axonal regeneration in the rat in adose-dependent manner following a sciatic nerve crush lesion (Gold etal., J. Neurosci. 15:7505-7516, 1995; Gold et al., Restor. Neurol.Neurosci. 6:287-296, 1994). FK506 was shown to stimulate neuriticoutgrowth in a rat pheochromocytoma cell line in aconcentration-dependent manner (Lyons et al., Proc. Natl. Acad. Sci. USA91:3191-3195, 1994).

[0003] Systemic administration of two synthetic FK506 analogs that bindFKBP-12 but that do not inhibit calcineurin activity (and which are notimmunosuppressants) increases the size of myelinated fibers (Steiner etal., Nature Medicine 3:1-8, 1997; Steiner et al., Proc. Natl. Acad. Sci.USA 94:2019-2024, 1997). U.S. Pat. No. 5,654,332 (Armistead et al.)discusses immunosuppressive FK506 analogs that bind FKBP12 and that aresaid to stimulate neurite outgrowth in the presence of NGF. It wasstated that the neurotrophic activity of these FKBP12 binding compounds“is directly related to their affinity for FKBP12 and their ability toinhibit FKBP12 rotomase activity” (id. at col. 7, lines 47-50).

[0004] It has been reported that androgens and estrogens stimulatefacial nerve regeneration in hamsters (Jones, “Androgenic enhancement ofmotor neuron regeneration,” In: Luine and Harding, eds., HormonalRestructuring of the Adult Brain, Ann. N.Y. Acad. Sci. 85:141-164, 1994;Tanzer and Jones, Exp. Neurol. 146:258-264, 1997).

SUMMARY OF THE INVENTION

[0005] I have discovered that geldanamycin and FK506 stimulate nerveregeneration via a common mechanism. Both compounds bind to polypeptidecomponents of steroid receptor complexes, hsp90 and FKBP52,respectively. These and other compounds that cause hsp90 dissociationfrom steroid receptor complexes or that block association of hsp90 withsteroid receptor complexes stimulate nerve cell growth and promote nerveregeneration. Such coupounds can act directly by binding to hsp90 (as inthe case of geldanamycin) or indirectly by binding to anotherpolypeptide in the steroid receptor complex (as in the case of FK506binding of FK3P52).

[0006] According to one aspect of the invention, pharmaceuticalcompositions are provided that include a nerve growth stimulating amountof a non-FKBP12-binding agent that binds to a polypeptide component of asteroid receptor complex other than the ligand (i.e., steroid hormone)binding portion thereof (such polypeptide components including, but notlimited to, hsp90 or FKBP52) and a pharmaceutically acceptableexcipient. Without limitation to any particular mechanism of action,binding of such agents to the polypeptide component likely causes hsp90dissociation from the complex or prevents hsp90 association with thecomplex. Nerve growth promoting agents according to the inventioninclude, but are not limited to non-FKBP12-binding FK506 analogs,benzoquinone ansamycins (e.g., geldanamycin and derivatives thereof),peptides that comprise a sequence of a selected polypeptide component ofthe complex at a site of interaction between the selected component andanother component of the complex, antibodies that bind a polypeptidecomponent of the steroid receptor complex, and combinations thereof.

[0007] According to another aspect of the invention, such pharmaceuticalcompositions include other active ingredients, including, but notlimited to, neurotrophic factor other than the nerve growth promotingagent (e.g., NGF, IGF-1, aFGF, bFGF, PDGF, BDNF, CNTF, GDNF, NT-3, NT4/5, or mixtures thereof), and a steroid ligand of the steroid receptorcomplex (e.g., estrogen and dexamethasone, as in the Examples below).

[0008] According to another aspect of the invention, a transection(severing of the nerve) of a periphal nerve or a spinal cord injury of amammal is treated by methods that include administering a nerve growthstimulating amount of a non-FKBP12-binding nerve growth promoting agentto the mammal and grafting to the peripheral nerve or spinal cord anallograft or an artificial nerve graft. In the case of a transectedperipheral nerve or spinal cord, the space between the transected endsof the peripheral nerve or spinal cord is preferably filled with amaterial such as collagen, methyl cellulose, etc., or a cell suspensionthat promotes nerve cell growth, such as Schwann cells and olfactory andsheathing cells. The nerve growth promoting agent can be includedtogether with with such filling materials.

[0009] According to another aspect of the invention, pharmaceuticalcompositions are provided that include a nerve growth stimulating amountof a non-FKBP12-binding FK506 analog that binds to FKBP52 and apharmaceutically suitable excipient.

[0010] According to another aspect of the invention, pharmaceuticalcompositions are provided that include a nerve growth stimulating amountof an agent that binds to hsp90 and causes hsp90 dissociation from asteroid receptor complex or prevents hsp90 association with the complexand a pharmaceutically suitable excipient.

[0011] According to another aspect of the invention, methods ofstimulating nerve cell growth in a mammal are provided that includeadministering a pharmaceutical composition as described herein.

[0012] According to another aspect of the invention, methods areprovided for stimulating growth of a nerve cell that include contactingthe nerve cell with a non-FKBP12-binding agent that binds to apolypeptide component of a steroid receptor complex other than thesteroid hormone binding portion thereof and causes hsp90 dissociationfrom the complex or blocks association of hsp90 with the complex.

[0013] According to another aspect of the invention, methods ofidentifying compounds that stimulate nerve cell growth are provided thatinclude the steps of assaying test compounds for binding to a componentof a steroid receptor complex other than the steroid hormone bindingportion thereof, and assaying the binding compounds for stimulation ofnerve cell growth. In addition, the binding compounds can be assayed foractivity in dissociation of hsp90 from the complex or blockingassociation of hsp90 with the complex.

[0014] The foregoing and various features and advantages of theinvention will become more apparent from the following detaileddescription and accompanying drawings.

BRIEF DESCRIPTION OF TEE DRAWINGS

[0015]FIG. 1 shows structures of FK506 (left) and a representative FK506analog, V-10,367 (right). The bracketed portion of FK506 represents thecalcineurin-binding domain, which is absent in V-10,367.

[0016] FIGS. 2-8 are histograms showing the stimulation of growth ofSH-SY5Y cells by geldanamycin and FK506 in the presence of NGF (10ng/mL) 168 hours after treatment.

[0017]FIG. 2: control cells (untreated).

[0018]FIG. 3: NGF only (10 ng/mL).

[0019]FIG. 4: geldanamycin (1 nM)+NGF (10 ng/mL).

[0020]FIG. 5: geldanamycin (10 nM)+NGF (10 ng/mL).

[0021]FIG. 6: FK506 (10 nM)+NGF (10 ng/mL).

[0022]FIG. 7: geldanamycin (1 nM)+FK506 (10 nM)+NGF (10 ng/mL).

[0023]FIG. 8: geldanamycin (10 nM)+FK506 (10 nM)+NGF (10 ng/mL).

[0024] FIGS. 9-15 are histograms showing the stimulation of growth ofSH-SY5Y cells by geldanamycin and FK506 in the presence of NGF (10ng/ml) 168 hours after treatment.

[0025]FIG. 9: control cells (untreated).

[0026]FIG. 10: NGF only (10 ng/mL).

[0027]FIG. 11: FK506 (1 nM)+NGF (10 ng/mL).

[0028]FIG. 12: FK506 (10 nM)+NGF (10 ng/mL)

[0029]FIG. 13: geldanamycin (0.1 nM)+NGF (10 ng/mL).

[0030]FIG. 14: geldanamycin (0.1 nM)+FK506 (1 nM)+NGF (10 ng/mL).

[0031]FIG. 15: geldanamycin (0.1 nM)+FK506 (10 nM)+NGF (10 ng/mL).

DETAILED DESCRIPTION

[0032] Members of the steroid/thyroid receptor family, including steroidreceptors such as the glucocorticoid and progesterone receptors, act asligand-inducible enhancers of specific gene expression.

[0033] Upon translation, steroid receptors are assembled intomultiprotein complexes. Steroid receptors exist in two states that arein dynamic equilibrium in the cell. An initial hormone receptor complexincludes the steroid receptor, hsp90, hsp70, and at least twoco-chaperones, p60 and Hip (p48). This initial complex is localized inthe cytosol and does not bind DNA or enhance specific genetranscription. The initial complex is in equilibrium with a metastable,nearly mature complex that lacks hsp90, p60, and Hip but includes p23and one of the three large immunophilins, FKBP52 (also called hsp56),FKBP54, or CyP40, and under some circumstances, hsp 70 (Smith et al.,Mol. Cell. Biol. 15:6804-6812, 1995; Dittmar et al., J. Biol. Chem.271:12833-12839, 1996). The nearly mature complex is competent to bindhormone; upon hormone binding, the receptor is released as an activetranscription factor (Smith et al., Mol. Cell. Biol. 15:6804-6812, 1995;Dittmar et al., J. Biol. Chem. 271:12833-12839, 1996). Steroid receptorcomplexes are constantly dissociating and reforming under physiologicalconditions.

[0034] Hsp90-mediated conformational maturation is required for nuclearhormone receptors to acquire or maintain a state competent to bindhormone (Smith et al., Mol. Cell. Biol. 15:6804-6812, 1995; Dittmar etal., J. Biol. Chem. 271:12833-12839, 1996). Geldanamycin, a benzoquinoneansamycin antibiotic, binds in a pharmacologically specific manner tohsp90 (Whitesell et al., Proc. Natl. Acad. Sci. USA 91:8324-8328, 1994)and prevents association of the p23 component of the heterocomplexassembly system with hsp90 (Johnson and Toft, Mol. Endocrinol.9:670-678, 1995). Geldanamycin permits dissociation of a steroidreceptor complex, permitting receptors to be transformed (i.e.,dissociated from hsp90), but blocks reassembly of the hormone-responsiveform of the complex, thereby preventing hormone activation andultimately resulting in the degradation of the hormone receptor.Geldanamycin blocks assembly of the progesterone receptor (PR) complex(Smith et al., Mol. Cell. Biol. 15:6804-6812, 1995) and of theglucocorticoid receptor (GR) complex (Czar et al., Biochem.36:7776-7785, 1997) at an intermediate stage of assembly where thehormone binding domain is not properly folded and therefore cannot bindsteroid. Geldanamycin also is known to act on estrogen and androgenhormone receptors (Smith et al., Mol. Cell. Biol. 15:6804-6812, 1995;Nair et al., Cell Stress and Chaperones 1:237-250, 1996). Transformationof GR and PR as measured either by 9S to 4S conversion or by acquisitionof DNA-binding activity is correlated with dissociation of steroidreceptors from hsp90 (see, e.g., Meshinchi et al., J. Biol. Chem.265:4863-4870, 1990; Kost et al., Mol. Cell. Biol. 9:3829-3838, 1989).

[0035] In addition to steroid receptors, other “substrate proteins” forhsp90 include v-erbA, dioxin receptor, sim, myoD1, E12, heat shockfactor, tumor promoter-specific binding protein, hepatitis B reversetranscriptase, p53 tumor suppressor mutant, various protein kinases(e.g., the tyrosine kinases v-src, c-src, v-fps, v-yes, v-fes, v-frg,c-frg, lck, Weel kinase, and sevenless PTK), heme-regulated eIF-2α,eEF-2 kinase, casein kinase II, v-raf, c-raf, Gag-Mil, MEK, PI-4 kinase,actin, tubulin, centrin, proteasome, and G_(βγ) complex (reviewed inPratt and Toft, Endocrine Rev. 18:306-360, 1997).

[0036] FKBP52 is a member of the FK506-binding class of immunophilins.Binding of FK506 to GR-associated FKBP52 caused increased nucleartranslocation of GR in response to dexamethasone and potentiation ofGR-mediated gene expression (Sanchez and Ning, METHODS: A Companion toMeth. Enzymol. 9:188-200, 1996). Dexamethasone-induced GR-specific geneexpression is also potentiated by cyclosporin A (CsA) (Renoir et al.,Proc. Natl. Acad. Sci. USA 92:4977-4981, 1995), rapamycin (Ning andSanchez, J. Biol. Chem. 268:6073-6076, 1993), and nonimmunosuppressiveanalogs of FK506 (e.g., 15-o-desmethyl FK520) or of CsA (e.g., CsH andSDZ220384) (Sanchez and Ning, METHODS: A Companion to Meth. Enzymol.9:188-200, 1996).

[0037] CyP40, rather than FKBP52, is the target for binding of CsA andits analogs (Sanchez and Ning, METHODS: A Companion to Meth. Enzymol.9:188-200, 1996). FKBP52 and CyP40 bind directly to hsp90, and CyP40competes for FKBP52 binding to hsp90 and vice versa. The immunophilinsbind hsp90 in a mutually exclusive fashion, leading to the formation ofseparate CyP40-hsp90 and FKBP52-hsp90 complexes (Ratajczak and Carrello,J. Biol. Chem. 271:2961-2965, 1996). Immunophilins such as FKBP52 andCyP40 and non-immunophilin proteins such as PP5, p60, and Mas70p, haveone or more tetratricopeptide repeat (TPR) domains (Ratajczak et al., J.Biol. Chem. 268:13187-13192, 1993) that bind to the TPR-binding domainof hsp90. The number of TPR domains in a protein appears to correlatewith its hsp90-binding affinity. Regions bordering the TPR domain alsoparticipate in binding, e.g., the acidic domain at the N-terminal end ofbovine CyP40 (residues 185-225) and FKBP52 (residues 232-271) and thecalmodulin binding region at the C-terminus of bovine CyP40 (Ratajczakand Carrello, J. Biol. Chem. 271:2961-2965, 1996). Binding of bothFKBP52 and Cy-P40 to hsp90 is competed by a purified fragment of humanCyP40 comprising its three TPR domains and by a fragment of rat PP5comprising its four TPR domains (reviewed in Pratt and Toft, EndocrineRev. 18:306-360, 1997).

[0038] In addition to multiple TPR binding domains, FKBP52 contains asequence (EDLTDDED in rabbit) that is retained with conservativereplacements in human and mouse. This negatively charged sequence iselectrostatically complementary to the receptor nuclear localizationsignals (e.g., the NL1 sequence RKTKKKIK of rat GR). An antibody raisedagainst the conserved negatively charged sequence impeded thedexamethasone-mediated shift of the GR into the nucleus (reviewed inPratt and Toft, Endocrine Rev. 18:306-360, 1997). It has also beenreported that antibodies directed against a conserved negatively-chargedsequence of FKBP52 impede dexamethasone-mediated cytophasmic-nucleartranslocation of GR (Czar et al., Mol. Endocrinol. 9:1549-1560, 1995).

[0039] The effects of FK506 and geldanamycin on nerve regenerationlikely result from the binding of these compounds to components ofsteroid receptor complexes, causing the dissociation of hsp90 from thesteroid receptor complex either directly (by binding to hsp90 orinterfering with the binding of hsp90 to the steroid receptor) orindirectly (by binding to a polypeptide such as FKBP52 that itself bindsto hsp90), or, alternatively, by preventing association of hsp90 withthe steroid receptor complex. However, interference with the ability ofhsp90 to complex with and perform its chaperone function for other hsp90substrate proteins may also be responsible for or contribute to theobserved stimulation of nerve regeneration by FK506 and/or geldanamycin.

[0040] Definitions and Methods

[0041] “Nerve Growth Promoting Agent” (NGPA).

[0042] A “nerve growth promoting agent” or NGPA is defined as asubstance that binds to a polypeptide component of a steroid receptorcomplex other than the steroid hormone binding portion thereof, suchcomponents including but not limited to hsp90 and FKBP52, and promotesnerve regeneration, without limitation to a particular mechanism ofaction. Preferably, the NGPA does not bind FKBP12 and isnon-immunosuppressive. NGPA include, but are not limited to,non-FKBP12-binding (“non-binding”) analogs of FK506; benzoquinoneansamycins, including geldanamycin, naturally occurring analogs ofgeldanamycin, including, but not limited to, herbimycin A and macbecin(DeBoer et al., J. Antibiot. (Tokyo) 23:442-447, 1970; Omura et al., J.Antibiot. (Tokyo) 32:255-261, 1979; Ono et al., Gann. 73:938-944, 1992),and derivatives thereof; peptides including an amino acid sequence of aparticular polypeptide component of a steroid receptor complex at a siteof interaction between that component and another component of thecomplex (such as the TPR domain), and antibodies that bind specificallyto polypeptide components of steroid receptor complexes, e.g.,anti-hsp90, anti-FKBP52, etc.) and interfere with the interaction of thebound polypeptide with another polypeptide in the steroid receptorcomplex.

[0043] “Steroid Receptor Complex” or “Steroid Hormone Receptor” and“Component” Thereof; “Transformation” “Activation”.

[0044] The term “steroid receptor complex” is intended to encompass amultiprotein complex associated with any steroid receptor, including,but not limited to, the progesterone receptor, glucocorticoid receptor,estrogen receptor, androgen receptor, and mineralocorticoid receptor. Apolypeptide “component” is a polypeptide other than the steroid hormonebinding portion of the steroid receptor complex, and preferably otherthan steroid receptor (particularly the steroid hormone binding portionthereof), such as hsp90, FKBP52, etc., that is part of a steroidreceptor complex.

[0045] The term “transformation” refers to the conversion of the 9Snon-DNA-binding form of a steroid receptor complex to the 4S DNA-bindingform. The term “activation” refers to the conversion of a steroidreceptor from a form that does not bind steroid to a steroid-bindingform.

[0046] Assays for Identifying NGPAs.

[0047] There are a number of well-known methods for assaying compoundsthat bind to hsp90, FKBP52, and other polypeptide components of asteroid receptor complex that can be used as an initial screen forcandidate compounds that stimulate nerve regeneration. Compounds cansubsequently be tested in vitro or in vivo for activity in stimulatingnerve regeneration.

[0048] For example, one may assay for the binding of a test compound toa polypeptide that is a component of a steroid receptor complex. Anassay for binding to hsp90 is described, for example, by Whitesell etal. (Proc. Natl. Acad. Sci. USA 91:8324-8328, 1994). Commercial hsp90(StressGen Biotechnologies, Victoria, BC) dissolved in 20 μg/mL of TNESVbuffer (50 mM Tris-HCl, pH 7.4/1% Nonidet P-40/2 mM EDTA/100 mM NaCl/1mM orthovanadate/1 mM phenylmethylsulfonyl fluoride/20 μg leupeptin permL/20 μg of aprotinin per ml) and the test compound are incubated for 45min at 4° C. with geldanamycin immobilized on a conventional solidsupport, e.g., geldanamycin-coupled agarose beads (Whitesell et al.,Proc. Natl. Acad. Sci. USA 91:8324-8328, 1994. The beads are then washedwith TNESV buffer and bound hsp90 is eluted by heating in reducingloading buffer and can be analyzed by SDS/PAGE and silver staining(Bio-Rad), for example. Alternatively, if the hsp90 is labeled, one canassay for bound label versus free label. Test compounds that competewith geldanamycin for binding to hsp90 inhibit the binding ofsolubilized hsp90 to the beads.

[0049] Similar assays can be performed to identify compounds that bindother steroid receptor complex polypeptide components. Binding to FKBP52can be assayed using recombinant FKBP52 (Peattie et al., Proc. Natl.Acad. Sci. USA 89:10974-10978, 1992) instead of hsp90 and immobilizedFK506 or FK506 analogs or hsp90. Binding to p23 can be assayed usingrecombinant human p23 (Johnson et al., Mol. Cell. Biol. 14:1956-1963,1994) and immobilized hsp90. Purified hsp70 and recombinant p60 (Dittmaret al., J. Biol. Chem. 271:12833-12839, 1996) are also available for usein such binding assays.

[0050] Immunoassays can also be performed using conventional immunoassaymethodologies and antibodies that are specific for steroid receptorcomplex components, e.g., antibodies against FKBP52 (Tai et al.,Biochem. 25:5269-5275, 1986), hsp90 (Sanchez et al., J. Biol. Chem.260:12398-12401, 1985; Catelli et al., EMBO J. 4:3131-3135, 1985; Schuhet al., J. Biol. Chem. 260:14292-14296, 1985), hsp70 (a serum that alsorecognizes hsp90 (Erhart et al., Oncogene 3:595-603, 1988), p23 (Johnsonet al., Mol. Cell. Biol. 14:1956-1963, 1994), etc.

[0051] A well-accepted qualitative assay for receptor transformation,which involves dissociation of hsp90 from the receptor complex, isconversion of a receptor complex to a state that binds polyanions suchas phosphocellulose (Kalimi et al., J. Biol. Chem. 250:1080-1086, 1975;Atger and Milgrom, Biochem. 15:4298-4304, 1976), ATP-Sepharose (Toft etal., J. Steroid Biochem. 7:1053-1059, 1976; Miller and Toft, Biochem.17:173-177, 1978), and carboxymethol-Sephadex (Milgrom et al., Biochem.12:5198-5205, 1973; Parchman and Litwack, Arch. Biochem. Biophys.183:374-382, 1977).

[0052] An in vitro assay for nerve cell growth (neurite outgrowth) isprovided in Example 1 below. In vivo assays for nerve regeneration arediscussed in, for example, Gold et al., Restor. Neurol. Neurosci.6:287-296, 1994; Gold et al., J. Neurosci. 15:7505-7516, 1995; Wang etal., J. Pharmacol. Exp. Therapeutics 282:1084-1093, 1997; Gold et al.,Exp. Neurol. 147:269-278, 1997; Gold et al., Soc. Neurosci. Abst.23:1131, 1997, which examine the effects of systematic administration ofa test compound on nerve regeneration and functional recovery followinga crush injury to the rat sciatic nerve. Briefly stated, the rightsciatic nerve of anaesthetized rats is exposed, and the nerve crushedtwice using forceps at the level of the hip. Following the sciatic nervecrush, the test compound is administered to the rats, e.g., bysubcutaneous injection or oral administration. Functional recovery isassessed by determining the number of days following nerve crush untilthe animal demonstrates onset of an ability to right its foot and moveits toes, and the number of days until the animal demonstrates anability to walk on its hind feet and toes. Nerve regeneration is alsoassessed by sampling tissues from the sciatic nerve at known (0.5 cm)distances from the crush site and examining the number of myelinatedfibers and the size of axons by light microscopy. The axons are alsoexamined by electron microscopy. Axonal areas of both myelinated andunmyelinated fibers are determined by tracing the axolemma using adigitizing tablet connected to a computer with appropriate software.Cumulative histograms are constructed from these data and mean valuesand standard errors are calculated to assess the effect ofadministration of the test compound on axonal areas.

[0053] “Geldanamycin Derivatives”.

[0054] “Geldanamycin derivatives” include well-known syntheticderivatives (Schnur et al., J. Med. Chem. 38:3813-3820, 1995; Schnur etal., J. Med. Chem. 38:3806-3812, 1995). Geldanamycin derivativespreferably have the carbamate group and ansa ring of geldanamycin, whichare necessary for activity (Schur et al., J. Med. Chem. 38:3806-3812,1995), including modifications at the C23 methoxy and C22 methyl groups(Stebbins et al., Cell 89:239-250, 1997). Geldanamycin derivatives arealso discussed in U.S. Pat. No. 5,3877,584, 4,261,989, and 3,987,035,and in Japanese Patent Applications 88041885, 56100766, and 89002593,for example.

[0055] “FK506 Analogs”.

[0056] As used herein, the term “FK506 analogs” refers to compounds thatare functionally analogous to FK506 in their ability to stimulateneuritic outgrowth. Such FK506 analogs, such as V-10,367, retain theFKBP12 binding domain but lack the structural components of the effectordomain (FIG. 1) and may either bind FKBP12 or be non-binding. V-10,367,for example, binds FKBP12 with high affinity (<1 nM) (Armistead et al.,Acta Crystallogr. 51:522-528, 1995).

[0057] There has been an intense effort to design compounds that arestructurally related to FK506 and that share the ability of FK506 toinhibit FKBP12 and thereby cause immunosuppression. See, for example:Bierer et al., Science 250:556-559, 1990; Van Duyne et al., Science252:839-842, 1991; Van Duyne et al., J. Mol. Biol. 229:105-124, 1993;Hauske et al., J. Med. Chem. 35:4284-4296, 1992; Holt et al., J. Am.Chem. Soc. 115:9925-9938, 1993; Holt et al., Bioorg. Med. Chem. Lett.3:1977-1980, 1993; Teague and Stocks, Bioorg. Med. Chem. Lett.3:1947-1950, 1993; Wang et al., Bioorg. Med. Chem. Lett. 4:1161-1166,1994; Yamashita et al., Bioorg. Med. Chem. Lett. 4:325-328, 1994; Stockset al., Bioorg. Med. Chem. Lett. 4:1457-1460, 1994; Goulet et al.,Perspect. Drug Disc. Design 2:145-162, 1994; Wilson et al., Acta Cryst.D51:511-S21, 1995; Armistead et al., Acta Cryst. D51:522-528, 1995; U.S.Pat. Nos. 5,192,773, 5,330,993, 5,516,797, 5,612,350, 5,614,547,5,622,970, 5,654,332; and published international patent applications WO92/00278, WO 92/04370, WO 92/19593, WO 92/21313, WO 94/07858, and WO96/40633.

[0058] FK506 analogs include, but are not limited to:

[0059] (1) Compounds represented by the formula I (see U.S. Pat. Nos.5,622,970, 5,516,797, 5,330,993, 5,192,773, and WO 92/00278 regardingsynthesis of these compounds, the disclosures of which are incorporatedherein by reference):

[0060] wherein A is O, NH, or N—(C1-C4 alkyl)

[0061] wherein B is hydrogen, CHL—Ar, (C1-C6)-straight or branchedalkyl, (C2-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl,(C5-C7)-cycloalkenyl or Ar substituted (C1-C6)-alkyl or (C2-C6)-alkenyl,or

[0062] wherein L and Q are independently hydrogen, (C1-C6)-straight orbranched alkyl or (C2-C6)-straight or branched alkenyl;

[0063] wherein T is Ar or substituted cyclohexyl with substituents atpositions 3 and 4 that are independently selected from the groupconsisting of hydrogen, hydroxyl, O—(C1-C4)-alkyl or O—(C2-C4)-alkenyland carbonyl;

[0064] wherein Ar is selected from the group consisting of 1-naphthyl,2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyland phenyl having one to three substituents that are independentlyselected from the group consisting of hydrogen, halo, hydroxyl, nitro,CF₃, (C1-C6)-straight or branched alkyl or (C2-C6)-straight or branchedalkenyl, O—(C1-C4)-straight or branched alkyl or O—(C2-C4)-straight orbranched alkenyl, O-benzyl, O-phenyl, amino and phenyl;

[0065] wherein D is U; E is either oxygen or CH—U, provided that if D ishydrogen, then E is CH—U or if E is oxygen, then D is not hydrogen;

[0066] wherein each U is independently selected from hydrogen,O—(C1-C4)-straight or branched alkyl or O—(C2-C4)-straight or branchedalkenyl, (C1-C6)-straight or branched alkyl or (C2-C6)-straight orbranched alkenyl, (C5-C7)-cycloalkyl or (C5-C7)-cycloalkenyl substitutedwith (C1-C4)-straight or branched alkyl or (C2-C4)-straight or branchedalkenyl, 2-indolyl, 3-indolyl, [(C1-C4)-alkyl or (C2-C4)-alkenyl]—Ar orAr;

[0067] wherein J is hydrogen or C1 or C2 alkyl or benzyl; K is(C1-C4)-straight or branched alkyl, benzyl or cyclohexylmethyl; orwherein J and K may be taken together to form a 5-7 memberedheterocyclic ring that may contain an oxygen (O), sulfur (S), SO or SO₂substituent therein; and

[0068] the stereochemistry at position 1 is R or S.

[0069] (2) Compounds represented by the formula II (see U.S. Pat. No.5,654,332, WO 94/07858, and WO 92/19593 for synthesis of thesecompounds, the disclosures of which are incorporated herein byreference):

[0070] wherein A′ is CH₂, oxygen, NH, or N—(C1-C4 alkyl);

[0071] wherein B′ and W are independently hydrogen, Ar′,(C1-C10)-straight or branched alkyl, (C2-C10)-straight or branchedalkenyl or alkynyl, (C5-C7)-cycloalkyl substituted (C1-C6)-straight orbranched alkyl, (C2-C6)-straight or branched alkenyl or alkynyl,(C5-C7)-cycloalkenyl substituted (C1-C6)-straight or branched alkyl,(C2-C6)-straight or branched alkenyl or alkynyl, or Ar′ substituted(C1-C6)-straight or branched alkyl, (C2-C6)-straight or branched alkenylor alkynyl wherein in each case, any one of the CH₂ groups of the alkyl,alkenyl, or alkynyl chains may be optionally replaced by a heteroatomselected from the group consisting of O, S, SO, SO₂, N, and NR, whereinR is selected from the group consisting of hydrogen, (C1-C4)-straight orbranched alkyl, (C2-C4)-straight or branched alkenyl or alkynyl, and(C1-C4) bridging alkyl wherein a bridge is formed between the nitrogenand a carbon atom of the heteroatom-containing chain to form a ring, andwherein the ring is optionally fused to an Ar′ group, or

[0072] wherein Q′ is hydrogen, (C1-C6)-straight or branched alkyl or(C2-C6)-straight or branched alkenyl or alkynyl;

[0073] wherein T′ is Ar or substituted 5-7 membered cycloalkyl withsubstituents at positions 3 and 4 that are independently selected fromthe group consisting of oxo, hydrogen, hydroxyl, O—(C1-C4)-alkyl, andO—(C2-C4)-alkenyl;

[0074] wherein Ar′ is a carboxcyclic aromatic group selected from thegroup consisting of phenyl, 1-naphthyl, 2-naphthyl, indenyl, azulenyl,fluorenyl, and anthracenyl; or a heterocyclic aromatic group selectedfrom the group consisting of 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, pyrrolyl, oxazolyl, thiazolyl,imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl,1,2,3-triazolyl, 1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, 1,3,5-triazinyl, 1,3,5-trithianyl, indolizinyl, indolyl,isoindolyl, 3H-indolyl, indolinyl, benzo[b]furanyl, benzo[b]thiophenyl,1H-indazolyl, benzimidazolyl, benzthiazolyl, purinyl, 4H-quinolizinyl,quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, and phenoxazinyl;

[0075] wherein Ar′ may contain one to three substituents that areindependently selected from the group consisting of hydrogen, halogen,hydroxyl, hydroxymethyl, nitro, trifluoromethyl, trifluoromethoxy,(C1-C6)-straight or branched alkyl, (C2-C6)-straight or branchedalkenyl, O—[(C1-C4)-straight or branched alkyl], O—[(C2-C4)-straight orbranched alkenyl], O-benzyl, O-phenyl, 1,2-methylenedioxy, amino,carboxyl, N—[(C1-C5)-straight or branched alkyl or (C2-C5)-straight orbranched alkenyl)carboxamides, N,N-di[(C1-C5)-straight or branched alkylor (C2-C5)-straight or branched alkenyl]carboxamides,N-morpholinocarboxamide, N-benzylcarboxamide,N-thiomorpholinocarboxamide, N-picolinoylcarboxamide, O—X,CH₂—(CH₂)_(q)—X, O—(CH₂)_(q)—X, (CH₂)_(q)—O—X, and CH═CH—X;

[0076] wherein X is 4-methoxyphenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,pyrazyl, quinolyl, 3,5-dimethylisoxazoyl, isoxazoyl, 2-methylthiazoyl,thiazoyl, 2-thienyl, 3-thienyl, or pyrimidyl; and a is 0-2;

[0077] wherein G is U′;

[0078] wherein M is either oxygen or CH-U′; provided that if G ishydrogen, then M is CH-U′ or if M is oxygen, then G is U′;

[0079] wherein U′ is hydrogen, O—[(C1-C4)-straight or branched alkyl] orO—[(C2-C4)-straight or branched alkenyl], (C1-C6)-straight or branchedalkyl or (C2-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl or(C5-C7)-cycloalkenyl substituted with (C1-C4)-straight or branched alkylor (C2-C4)-straight or branched alkenyl, [(C1-C4)-alkyl or(C2-C4)-alkenyl]-Y or Y;

[0080] wherein Y is selected from the group consisting of phenyl,1-naphthyl, 2-naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl,2-pyrrolinyl, 3-pyrrolinyl, pyrolidinyl, 1,3-dioxolyl, 2-imidazolinyl,imidazolidinyl, 2H-pyranyl, 4H-pyranyl, piperidyl, 1,4-dioxanyl,morpholinyl, 1,4-dithianyl, thiomorpholinyl, piperazinyl, quinuclidinyl,and heterocyclic aromatic groups as defined for Ar′ above;

[0081] wherein Y may contain one to three substituents that areindependently selected from the group consisting of hydrogen, halogen,hydroxyl, hydroxymethyl, nitro, trifluoromethyl, trifluoromethoxy,(C1-C6)-straight or branched alkyl, (C2-C6)-straight or branchedalkenyl, O—[(C1-C4)-straight or branched alkyl], O—[(C2-C4)-straight orbranched alkenyl], O-benzyl, O-phenyl, 1,2-methylenedioxy, amino, andcarboxyl;

[0082] wherein J′ is hydrogen, (C1-C2) alkyl or benzyl; wherein K is(C1-C4)-straight or branched alkyl, benzyl or cyclohexylmethyl, orwherein J′ and K may be taken together to form a 5-7 memberedheterocyclic ring that may contain a heteroatom selected from the groupconsisting of O, S, SO and SO₂;

[0083] wherein m is 0-3; and

[0084] wherein the stereochemistry at position 1 is R or S and thestereochemistry at position 2 is R or S.

[0085] (3) Compounds represented by the formula III (see Armistead etal., Acta Cryst. D51:522-528, 1995, including a discussion of selectionof R and of the synthesis of these compounds, the disclosure of which isincorporated herein by reference):

[0086] (4) Compounds represented by the formula IV (see WO 92/21313,including a discussion of the synthesis of these compounds, thedisclosure of which is incorporated herein by reference):

[0087] wherein A is CH₂, oxygen, NH or N—(C1-C4 alkyl);

[0088] wherein B and D are independently Ar, hydrogen, (C1-C6)-straightor branched alkyl, (C1-C6)-straight or branched alkenyl,(C1-C6)-straight or branched alkyl or alkenyl that is substituted with a(C5-C7)-cycloalkyl, (C1-C6)-straight or branched alkyl or alkenyl thatis substituted with a (C5-C7)-cycloalkenyl, or Ar substituted(C1-C6)-straight or branched alkyl or alkenyl, wherein, in each case,one or two of the CH₂ groups of the alkyl or alkenyl chains may contain1-2 heteroatoms selected from the group consisting of oxygen, sulfur, SOand SO₂ in chemically reasonable substitution patterns, or

[0089] provided that both B and D are not hydrogen;

[0090] wherein Q is hydrogen, (C1-C6)-straight or branched alkyl or(C1-C6)-straight or branched alkenyl;

[0091] wherein T is Ar of substituted 5-7 membered cycloalkyl withsubstituents at positions 3 and 4 that are independently selected fromthe group consisting of hydrogen, hydroxyl, O—(C1-C4)-alkyl,O—(C1-C4)-alkenyl and carbonyl;

[0092] wherein Ar is selected from the group consisting of phenyl,1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, monocyclic and bicyclic heterocyclicring systems with individual ring sizes being 5 or 6 that may contain ineither or both rings a total of 1-4 heteroatoms independently selectedfrom O, N and S; wherein Ar may contain one to three substituents thatare independently selected from the group consisting of hydrogen, halo,hydroxyl, nitro, trifluoromethyl, trifluoromethoxy, (C1-C6)-straight orbranched alkyl, (C2-C6)-straight or branched alkenyl, O—(C1-C4)-straightor branched alkyl, O—(C2-C4)-straight or branched alkenyl, O-benzyl,O-phenyl, 1,2-methylenedioxy, amino, carboxyl and phenyl;

[0093] wherein E is (C1-C6)-straight or branched alkyl, (C1-C6)-straightor branched alkenyl, (C5-C7)-cycloalkyl, (C5-C7)-cycloalkenylsubstituted with (C1-C4)-straight or branched alkyl or (C1-C4)-straightor branched alkenyl, [(C2-C4)alkyl or (C2-C4)-alkenyl)]-Ar or Ar (Ar asdescribed above);

[0094] wherein J is hydrogen or C1 or C2 alkyl or benzyl; K is(C1-C4)-straight or branched alkyl, benzyl or cyclohexylmethyl; orwherein J and K may be taken together to form a 5-7 memberedheterocyclic ring that may contain an oxygen, sulfur, SO or SO₂substituent therein; and;

[0095] wherein n is 0-3; and

[0096] wherein the stereochemistry at position 1 is R or S and thestereochemistry at position 2 being R or S.

[0097] (5) Compounds represented by the formula V (see WO 92/04370,including a discussion of the synthesis of these compounds, thedisclosure of which is incorporated herein by reference):

[0098] wherein A is NH, O, S, or CH;

[0099] wherein if A is NH, O, or S, B is PCO— or POCO—, where P is aC1-C6 straight or branched alkyl or alkenyl group, a C5-C6 cycloalkyl orcycloalkenyl, or a methyl substituted with a C5-C6 cycloalkyl, C5-C6cycloalkenyl, phenyl, 1-naphthyl, 2-naphthyl, 9-fluorenyl, or1-adamantyl;

[0100] wherein if A is CH, then B is connected via a trans double bondand is a C2-C4 straight or branched alkyl or alkenyl group, or is amethyl or ethyl substituted with either a C5-C6 cyclic alkyl group orAr, where Ar is selected from the group consisting of 1-naphthyl,2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, phenyl and phenyl having one tothree substituents that are independently selected from the groupconsisting of: hydroxyl, halo, nitro, CF₃, C1-C4 straight or branchedalkyl or alkenyl, O—(C1-C4) straight or branched alkyl or alkenyl, andAr, where Ar is selected from the group consisting of 1-naphthyl,2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, phenyl and phenyl having one tothree substituents that are independently selected from the groupconsisting of: hydroxyl, halo, nitro, CF₃, C1-C4 straight or branchedalkyl or alkenyl, O—(C1-C4) straight or branched alkyl or alkenyl;wherein no more than two Ar groups may be linked together;

[0101] wherein D is hydrogen, C1-C4 straight or branched alkyl oralkenyl, hydroxy, tert-butyloxy, benzyloxy, 4-benzyloxyphenyl,cyclohexyl, —(CH₂)_(n)—CO₂—Q, where n=0 or 1 and Q is methyl, ethyl,i-propyl, t-butyl, benzyl, 1-naphthyl, 2-naphthyl, of cyclohexyl; or Arwhere Ar is selected from the group consisting of 1-naphthyl,2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, phenyl and phenyl having one tothree substituents that are independently selected from the groupconsisting of: hydroxyl, halo, nitro, CF₃, C1-C4 straight or branchedalkyl or alkenyl, O—(C1-C4) straight or branched alkyl or alkenyl, andAr, where Ar is selected from the group consisting of 1-naphthyl,2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, phenyl and phenyl having one tothree substituents that are independently selected from the groupconsisting of: hydroxyl, halo, nitro, CF₃, C1-C4 straight or branchedalkyl or alkenyl, O—(C1-C4) straight or branched alkyl or alkenyl;wherein no more than two Ar groups may be linked together;

[0102] wherein E and K are independently hydrogen or methyl;

[0103] wherein G is either methyl or ethyl; J is hydrogen, C1-C6straight or branched alkyl or alkenyl, C6-C6 cycloalkyl or cycloalkenyl,sulfhydryl, hydroxy, phenyl, 3-indolyl, or benzyl; wherein G and J maybe connected by a bond to form a cycle of 5 or 6 members;

[0104] wherein L is O or an α-amino acid residue attached via theα-nitrogen, and selected from the group consisting of: alanine,2-aminobutyric acid, valine, norvaline, leucine, norleucine, isoleucine,phenylalanine, cyclohexylalanine, tryptophan, 1-naphthylalanine,2-naphthylalanine, threonine (side chain benzyl or tert-butyl ether),methionine, or serine (side chain benzyl or tert-butyl ether);

[0105] wherein if L is O, then M is C1-C6 straight or branched alkyl oralkenyl, or —(CH₂)_(n)—Ar, where n=1-6 and Ar is selected from the groupconsisting of: 1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl,phenyl and phenyl having one to three substituents that areindependently selected from the group consisting of: hydroxyl, halo,nitro, CF₃, C1-C4 straight or branched alkyl or alkenyl, O—(C1-C4)straight or branched alkyl or alkenyl, and Ar, wherein Ar is selectedfrom the group consisting of: 1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl2-thienyl, phenyl and phenyl having one to three substituents that areindependently selected from the group consisting of: hydroxyl, halo,nitro, CF₃, C1-C4 straight or branched alkyl or alkenyl, O—(C1-C4)straight or branched alkyl or alkenyl; wherein no more than two Argroups may be linked together;

[0106] wherein if L is an amino acid, then M is O—(C1-C4) straight orbranched alkyl, O-benzyl, NH-Phenyl, or NH-4-nitrophenyl and is attachedto the amino acid carbonyl;

[0107] the stereochemistry at all positions being R or S, and preferablythe stereochemistry is S at L if L is an a-amino acid, and at thosepositions marked with asterisks; however, when J is sulfhydryl, thepreferred stereochemistry of the asterisked position immediatelyadjacent to the nitrogen is R.

[0108] (6) Compounds represented by the formula VI (see WO 96/40633,including a discussion of the synthesis of these compounds, thedisclosure of which is incorporated herein by reference):

[0109] wherein, R1 is selected from the group consisting of a C1-C9straight or branched chain alkyl or alkenyl group optionally substitutedwith C3-C8 cycloalkyl, C3 or C5 cycloalkyl, C5-C7 cycloalkenyl, or Ar1,where said alkyl, alkenyl, cycloalkyl or cycloalkenyl groups may beoptionally substituted with C1-C4 alkyl, C1-C4 alkenyl, or hydroxy,where Ar1 is selected from the group consisting of 1-naphthyl,2-naphthyl, 2-indolyl, 3-indolyl, 2-furyl, 3-furyl, 2-thiazolyl,2-thienyl, 3-thienyl, 2-, 3-, 4-pyridyl, and phenyl, having one to threesubstituents that are independently selected from the group consistingof hydrogen, halo, hydroxyl, nitro, trifluoromethyl, C1-C6 straight orbranched alkyl or alkenyl, C1-C4 alkoxy or C1-C4 alkenyloxy, phenoxy,benzyloxy, and amino;

[0110] wherein X is selected from the group consisting of oxygen,sulfur, methylene (CH₂), or H₂;

[0111] wherein Y is selected from the group consisting of oxygen or NR₂,where R₂ is hydrogen or C1-C6 alkyl; and

[0112] wherein Z is selected from the group consisting of C2-C6 straightor branched chain alkyl or alkenyl, wherein the alkyl chain issubstituted in one or more positions with Ar₁ as defined above, C3-C8cycloalkyl, cycloalkyl connected by a C1-C6 straight or unbranched alkylor alkenyl chain and Ar₂, where Ar₂ is selected from the groupconsisting of 2-indolyl, 3-indolyl, 2-furyl, 3-furyl, 2-thiazolyl,2-thienyl, 3-thienyl, 2-, 3-, or 4-pyridyl, and phenyl, having one tothree substituents that are independently selected from the groupconsisting of hydrogen, halo, hydroxyl, nitro, trifluoromethyl, C1-C6straight or branched alkyl or alkenyl, C1-C4 alkoxy or C1-C4 alkenyloxy,phenoxy, benzyloxy, and amino;

[0113] wherein Z may also be the fragment:

[0114] where

[0115] R₃ is selected from the group consisting of straight or branchedalkyl C1-C8 optionally substituted with C3-C8 cycloalkyl, or Ar₁ asdefined above, and unsubstituted Ar₁;

[0116] X₂ is O or NR₅, where R₅ is selected from the group consisting ofhydrogen, C1-C6 straight or branched alkyl and alkenyl;

[0117] R₄ is selected from the group consisting of phenyl, benzyl, C1-C5straight or branched alkyl or alkenyl, and C1-C5 straight or branchedalkyl or alkenyl substituted with phenyl;

[0118] wherein the stereochemistry at position 1 is R or S.

[0119] Also encompassed are pharmaceutically acceptable derivatives ofthe FK506 analogs, including, but not limited to, any pharmaceuticallyacceptable salt, ester, salt of an ester, or any other derivative which,upon administration to a patient, is capable of providing directly orindirectly a non-binding FK506 analog or a metabolite or residue thereofthat has the desired neurotrophic activity. Included within the scope ofthe invention are enantiomers, the racemic form, and diastereoisomericmixtures. Enantiomers and diastereoisomers can be separated byconventional methods.

[0120] Formulae I-VI above represent compounds that have a wide range ofbinding affinities for FKBP12. The mechanism for neurotrophic activityof FK506 presented herein indicates that the effectiveness of FK506 andFK506 analogs in stimulating nerve cell growth is unrelated to theirability to bind FKBP12. Instead, their effectiveness in stimulatingnerve cell growth relates to ability of such compounds to bind FKBP52and subsequently interfere with the interaction of FKBP52 and hsp90 in asteroid receptor complex, e.g., by competing for FKBP52 binding tohsp90, altering the conformation of FKBP52, etc.

[0121] A “non-binding FK506 analog” is defined as an FK506 analog thatdoes not bind to FKBP12. Preferably, such FK506 analogs bind FKBP12 withan apparent K_(d) of greater than 10 μM as measured using well-knownassays, and preferably greater than 30 μM, and more preferably greaterthan 100 μM Values for the apparent K_(d) can be determined, forexample, by a competitive LH-20 binding assay performed as described,for example, in Harding et al., Nature 341:758-760, 1989 (using32-[1-¹⁴C]-benzoyl FK506 as a reporting ligand; Siekierka et al., Nature341:755-757, 1989, using [³H]dihydro-FK506 as a reporting ligand); andU.S. Pat. No. 5,654,332.

[0122] Alternatively, a “non-binding FK506 analog” is defined as anFK506 analog that does not significantly inhibit FKBP12 rotomaseactivity when administered to a patient at dosage levels of about 0.01to about 100 mg/kg body weight/day. Assays for inhibition of FKBP12rotamase activity are described in Harding et al. (Nature 341:758-760,1989), Siekierka et al., Nature 341:755-757, 1989, and U.S. Pat. No.5,654,332, for example. The assays of Harding et al. and Siekierka etal. employ a reaction mixture that includes the cis form ofN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, FKBP12, a test compound, andchymotrypsin, and spectrophotometrically measure the release ofp-nitroanilide as a result of isomerization of the substrate.

[0123] Non-binding FK506 analogs are non-immunosuppressive, as can bedemonstrated by well-known assays, e.g., as discussed in U.S. Pat. No.5,516,797, WO 92/21313, WO 92/19593, and WO 92/04370.

[0124] Non-binding FK506 analogs can be used in the form of saltspreferably derived from inorganic or organic acids and bases, including,but not limited to: acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.Base salts include, but are not limited to, ammonium salts, alkali metalsalts (such as sodium and potassium salts), alkaline earth metal salts(such as calcium and magnesium salts), salts with organic bases (such asdicyclohexylamine salts), N-methyl-D-glucamine, and salts with aminoacids (such as arginine, lysine, etc.). Basic nitrogen-containing groupscan be quaternized, e.g., with such agents as lower alkyl halides (suchas methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides),dialkyl sulfates (such as dimethyl, diethyl, dibutyl, an diamylsulfates), long-chain halides (such as decyl, lauryl, myristyl, andstearyl chlorides, bromides, and iodides), aralkyl halides (such asbenzyl and phenethyl bromides), etc. Water or oil-soluble or dispersibleproducts are produced thereby.

[0125] Non-binding FK506 analogs can be modified by appendingappropriate functionalities by well-known methods to enhance selectedbiological properties, including increasing penetration of the analogsinto a given cellular compartment (e.g., blood, lymphatic system,central nervous system, etc.), increase oral availability, increasesolubility to permit administration by injection, alter metabolism, andalter rate of excretion, for example.

[0126] Preferably, the non-binding FK506 analogs have a molecular weightbelow about 750 atomic mass units (a.m.u.) (as the parent compound,although the salts of such compounds can have higher molecular weights).

[0127] “Effective Amount” or “Nerve Growth Stimulating Amount.”

[0128] An “effective amount” or a “nerve growth stimulating amount” of acomposition according to the invention is an amount sufficient toachieve a statistically significant promotion of nerve cell growth orregeneration compared to a control. Nerve cell growth or nerveregeneration can be readily assessed using an in vitro assay, e.g., theassay described in the Examples below. Alternatively, nerve cell growthor regeneration can be determined in an in vivo assay or by direct orindirect signs of nerve cell growth and regeneration in a patient.Preferably, the increase in nerve cell growth or regeneration is atleast 10%, preferably at least 30%, and most preferably 50% or morecompared to a control. Preferred dosage levels are between about 0.1 toabout 400 mg/kg per day of the FK506 analog for subcutaneous delivery.For oral administration, preferred dosage levels are between about 0.01to about 40 mg/kg/day.

[0129] Therapeutic and Prophylactic Uses

[0130] Pharmaceutical compositions according to the invention can beperiodically administered to a mammalian patient (e.g., a humanpatient), in need of such treatment, to promote neuronal regenerationand functional recovery and to stimulate neurite outgrowth and therebyto treat various neuropathological states, including damage toperipheral nerves and the central nervous system caused by physicalinjury (e.g., spinal cord injury and trauma, sciatic or facial nervelesion or injury), disease (e.g., diabetic neuropathy), cancerchemotherapy (e.g., by vinca alkaloids and doxorubicin), brain damageassociated with stroke and ischemia associated with stroke, andneurological disorders including, but not limited to, various peripheralneuropathic and neurological disorders related to neurodegenerationincluding, but not limited to: trigeminal neuralgia, glossopharyngealneuralgia, Bell's palsy, myasthenia gravis, muscular dystrophy,amyotrophic lateral sclerosis, progressive muscular atrophy, progressivebulbar inherited muscular atrophy, herniated, ruptured or prolapsedvertebral disk syndromes, cervical spondylosis, plexus disorders,thoracic outlet destruction syndromes, peripheral neuropathies such asthose caused by lead, acrylamides, gamma-diketones (glue-sniffer'sneuropathy), carbon disulfide, dapsone, ticks, porphyria, Gullain-Barresyndrome, Alzheimer's disease, Parkinson's disease, and Huntington'schorea.

[0131] In addition, pharmaceutical compositions according to the presentinvention display a wide range of other therapeutic or prophylacticproperties, including, treatment of stroke (see, e.g., Sharkey andButcher, Nature 371:336-339, 1994, Vagita et al., Life Sciences59:1643-1650, 1996; Tokime et al., Neurosci. Lett. 206:81-84, 1996;Drake et al., Acta. Physiol. Scand. 158:155-159, 1996; and Kuroda etal., Neurosci. Res. Comm. 19:83-90, 1996), AIDS dementia (see, e.g.,Dawson and Dawson, Adv. Neuroimmunol. 4:167-173, 1994; and Sekigawa etal., J. Clin. Immunol. 15:312-317, 1995); hair growth (Yamamoto et al.,J. Investig. Dermatol. S 102:160-164, 1994; Jiang et al., J. Investig.Dermatol. 104:523-525, 1995); and connective tissue disorders (see e.g.,Steinmann et al., J. Biol. Chem. 266:1299-1303, 1991), and as a malecontraceptive (see e.g., Hisatomi et al., Toxicology 109:75-83, 1996).

[0132] A transection of a periphal nerve or a spinal cord injury can betreated by administering a nerve growth stimulating amount of anon-FKBP12-binding nerve growth promoting agent to the mammal andgrafting to the peripheral nerve or spinal cord an allograft (Osawa etal., J. Neurocytol. 19:833-849, 1990; Buttemeyer et al., Ann. PlasticSurgery 35:396-401, 1995) or an artificial nerve graft (Madison andArchibald, Exp. Neurol. 128:266-275, 1994; Wells et al., Exp. Neurol.146:395-402, 1997). The space between the transected ends of theperipheral nerve or spinal cord is preferably filled with a non-cellulargap-filling material such as collagen, methyl cellulose, etc., or cellsuspensions that promote nerve cell growth, such as Schwann cells (Xu etal., J. Neurocytol. 26:1-16, 1997), olfactory cells, and sheathing cells(Li et al. Science 277:2000-2002, 1997). The nerve growth promotingagent can be included together with with such cellular or non-cellulargap-filling materials.

[0133] Pharmaceutical Formulations

[0134] Pharmaceutical formulations according to the present inventionencompass formulations comprising (1) an amount (for example, a unitdosage) of an NGPA together with (2) one or more well-known non-toxicpharmaceutically acceptable excipients, including carriers, diluents,and/or adjuvants, and optionally (3) one or more biologically activeingredients. Standard pharmaceutical formulation techniques are used,such as those disclosed in Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa. (latest edition).

[0135] A pharmaceutical formulation according to the invention includesone or more NGPAs and can also include, for example, one or more otherbiologically active ingredients, including, but not limited to FK506 oran FKBP12-binding FK506 analogs or one or more other neurotrophicagents, including, for example, NGF, IGF-1, aFGF, bFGF, PDGF, BDNF,CNTF, GDNF, NT-3, and NT 4/5; and so on.

[0136] It is preferred that the pharmaceutical formulation includes anamount of a neurotrophic agent(s), preferably NGF, such that the patientreceives a dosage of between about 0.01 to 100 μg/kg body weight/day ofthe neurotrophic agent, or that the neurotrophic agent be administeredseparately, e.g., in separate single or multiple dosage forms,preferably concurrently, consecutively, or within less than about fivehours of each other.

[0137] The compositions can be in the form of tablets, capsules,powders, granules, lozenges, liquid or gel preparations, such as oral,topical, or sterile parenteral solutions or suspensions (e.g., eye orear drops, throat or nasal sprays, etc.), transdermal patches, and otherforms known in the art.

[0138] Such pharmaceutical compositions can be administered systemicallyor locally in any manner appropriate to the treatment of a givencondition, including orally, parenterally, rectally, nasally, buccally,vaginally, topically, optically, by inhalation spray, or via animplanted reservoir. The term “parenterally” as used herein includes,but is not limited to subcutaneous, intravenous, intramuscular,intrasternal, intrasynovial, intrathecal, intrahepatic, intralesional,and intracranial administration, for example, by injection or infusion.For treatment of the central nervous system, the pharmaceuticalcompositions preferably readily penetrate the blood-brain barrier whenperipherally administered or are administered intraventricularly.

[0139] Pharmaceutically acceptable carriers include, but are not limitedto, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins(such as human serum album n), buffers (such as phosphates), glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat, for example.

[0140] Tablets and capsules for oral administration can be in a formsuitable for unit dose presentation and can contain conventionalpharmaceutically acceptable excipients. Examples of these includebinding agents such as syrup, acacia, gelatin, sorbitol, tragacanth, andpolyvinylpyrrolidone; fillers such as lactose, sugar, corn starch,calcium phosphate, sorbitol, or glycine; tableting lubricants, such asmagnesium stearate, talc, polyethylene glycol, or silica; disintegrants,such as potato starch; and dispersing or wetting agents, such as sodiumlauryl sulfate. The tablets can be coated according to methods wellknown in normal pharmaceutical practice. Oral liquid preparations can bein the form of, for example, aqueous or oily suspensions, solutions,emulsions, syrups or elixirs, or can be presented as a dry product forreconstitution with water or other suitable vehicle before use. Suchliquid preparations can contain conventional additives such assuspending agents, e.g., sorbitol, syrup, methyl cellulose, glucosesyrup, gelatin, hydrogenated edible fats, emulsifying agents, e.g.,lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles(including edible oils), e.g., almond oil, fractionated coconut oil,oily esters such as glycerine, propylene glycol, or ethyl alcohol;preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid,and, if desired, conventional flavoring or coloring agents.

[0141] Pharmaceutical compositions according to the present inventioncan also be administered parenterally in a sterile aqueous or oleaginousmedium. The composition can be dissolved or suspended in a non-toxicparenterally-acceptable diluent or solvent, e.g., as a solution in1,3-butanediol. Adjuvants such as local anesthetics, preservatives, andbuffering agents can also be dissolved in the vehicle. Commonly usedvehicles and solvents include water, physiological saline, Hank'ssolution, Ringer's solution, and sterile, fixed oils, includingsynthetic mono- or di-glycerides, etc. Fatty acids, such as oleic acidand its glyceride derivatives are useful in the preparation ofinjectables, as are natural pharmaceutically acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant, such as Ph. Helv or a similar alcohol.

[0142] For topical application, the drug may be made up into a solution,suspension, cream, lotion, ointment in a suitable aqueous or non-aqueousvehicle. Additives may also be included, e.g., buffers such as sodiummetabisulphite or disodium edeate; preservatives such as bactericidaland fungicidal agents, including phenyl mercuric acetate or nitrate,benzalkonium chloride or chlorhexidine, and thickening agents, such ashypromellose.

[0143] The dosage unit involved depends, for example, on the conditiontreated, nature of the formulation, nature of the condition, embodimentof the claimed pharmaceutical compositions, mode of administration, andcondition and weight of the patient. Dosage levels on the order of about0.1 to about 400 mg/kg per day of the active ingredient are useful inthe treatment of the conditions listed above.

[0144] The invention will be better understood by reference to thefollowing examples, which are intended to merely illustrate the bestmode now known for practicing the invention. The scope of the inventionis not to be considered limited thereto, however.

EXAMPLES

[0145] FK506 and Geldanamycin Promote Nerve Regeneration by a CommonMechanism

[0146] Materials and Methods

[0147] Cell Cultures.

[0148] SH-SY5Y human neuroblastoma cells were maintained in DMEM medium(GIBCO) supplemented with 10% fetal calf serum (SIGMA), 50 IU/mLpenicillin, and 50 mg/mL streptomycin (GIBCO) at 37° C. in 7% CO₂. Cellswere plated in six-well plates at 1×10⁶ cells/well and treated with 0.4mM aphidicolin (SIGMA). At five days, cells were washed, treated withnerve growth factor (NGF) (Boehringer Mannheim, Indianapolis, Ind.) at10 ng/mL (to induce process outgrowth) in the presence or absence ofFK506 (1 and 10 nM) (Calbiochem-Novabiochem Int'l., La Jolla, Calif.)and/or geldanamycin (0.1, 1, and 10 nM) (Calbiochem-Novabiochem, LaJolla, Calif.). Media was changed at 96 hours and replaced with freshmedia with the compounds (NGF plus FK506 and/or geldanamycin) for anadditional 72 hours (total time, 168 hours). The top 50% of axonallengths were selected for statistical analysis. All experiments were runin duplicate wells and repeated at least twice for reproducibility.

[0149] Light Morphometry of Neurite Lengths.

[0150] For analysis of process length, cells 20 fields per well) wererandomly photographed at 72 and 168 hours. Neurite lengths were measuredon photographic prints using a Houston Instrument HI-PAD digitizingtablet connected to an IBM XT computer with appropriate software(Bioquant IV, R&M Biometrics, Nashville, Tenn.); only those processesgreater than two times the cell body length were measured. Data fromidentically treated wells were not different and were thereforecombined. Mean values and histograms were constructed from these data.Histograms were compared using a Mann-Whitney U test, which makes noassumptions about the shape of the distribution.

[0151] Preparation of FK506 and Geldanamycin.

[0152] FK506 (mol. wt. 822) and geldanamycin (mol. wt. 561) weredissolved in DMEM medium.

[0153] Results

[0154] In a first set of experiments, SH-SY5Y neuroblastoma cells wereplated in 6-well plates with DMEM plus 15% FCS and differentiated withNGF (10 ng/ml). The effects of various concentrations of geldanamycinand FK506 on neurite growth, alone and in combination, were tested. Themean lengths of neuritic processes of untreated and treated cells areshown in Table 1.

[0155] The cells developed long axonal-like processes upon exposure toNGF (10 ng/mL) as measured at 168 hours after treatment. NGF more thandoubled the mean length of the processes compared to untreated cells(compare FIGS. 1 and 2). An even greater increase in the length of theprocesses was observed when the cells were exposed to geldanamycin at 1nM in the presence of NGF (FIG. 3). However, geldanamycin at 10 nM hadno effect (FIG. 4). FK506 (10 nM) stimulated neurite outgrowth in thepresence of NGF (FIG. 5) to a greater degree than geldanamycin at either1 nM or 10 nM. In combination with FK506 (10 nM), geldanamycin (FIG. 6,1 nM; FIG. 7, 10 nM) inhibited the effect of FK506 in aconcentration-dependent fashion.

[0156] In a second set of experiments, lower concentrations ofgeldanamycin and FK506 were tested, alone and in combination. The meanlengths of neuritic processes of untreated and treated cells are shownin Table 2. NGF-treated cells (FIG. 9) had mean neuritic lengths morethan double the mean length of untreated control cells (FIG. 8). Nervegrowth stimulation was observed with FK506 (1 nM, FIG. 10; 10 nM, FIG.11) in the presence of NGF and geldanamycin (0.1 nM, FIG. 12) in thepresence of NGF. The lower concentration of FK506 (1 nM) was moreeffective in stimulating neurite outgrowth than the higher concentration(10 nM), and geldanamycin at 0.1 nM was even more effective instimulating neurite outgrowth than FK506 at either 1 nM or 10 nM.Combined treatment with geldanamycin (0.1 nM) and FK506 (1 nM, FIG. 13;10 nM, FIG. 14) in the presence of NGF showed that the effects ofgeldanamycin and FK506 were additive, particularly at the lower FK506concentration (FIG. 13).

[0157] Geldanamycyin and FK506 each stimulate neurite outgrowth in aconcentration-dependent fashion. Taken together, the similar effects ofgeldanamycin and FK506 in stimulating neurite outgrowth, their additiveeffects at low concentrations, and their inhibitory effects at highconcentrations (like high concentrations of either compound alone),demonstrate that the two compounds act on nerve cells via a commonmechanism. That mechanism likely involves an interaction of bothcompounds with components of steroid receptor complexes. FKBP12 does notappear to play a role in the stimulation of neurite outgrowth by eithergeldanamycin or FK506.

[0158] In further experiments, we have found that estrogen (10 nM) anddexamethasone (10 nM) increased neurite outgrowth (without NGF) ofSH-SY5Y cells and produced an additive effect on neurite outgrowth(neurotrophic action) with FK506 (10 nM).

[0159] This invention has been detailed both by example and by directdescription. It should be apparent that one having ordinary skill in therelevant art would be able to surmise equivalents to the invention asdescribed in the claims which follow but which would be within thespirit of the foregoing description. Those equivalents are to beincluded within the scope of this invention. TABLE 1 Mean Length of Top50% of Neuritic Processes of SH-SY5Y 168 Hours After Treatment withGeldanamycin (1 nM or 10 nM) and/or FK506 (10 nM) in the Presence of NGFTreatment Mean Length (μM) S.E.M. Untreated 41.61 1.25 NGF (10 ng/mL)53.99 2.26 Geldanamycin (1 nM) + 64.00 2.36 NGF (10 ng/mL) Geldanamycin(10 nM) + 54.81 2.10 NGF (10 ng/mL) FK506 (10 nM) + 77.82 2.70 NGF (10ng/mL) Geldanamycin (1 nM) + 72.63 2.01 FK506 (10 nM) + NGF (10 ng/mL)Geldanamycin (10 nM) + 67.41 1.67 FK506 (10 nM) + NGF (10 ng/mL)

[0160] TABLE 2 Mean Length of Top 50% of Neuritic Processes of SH-SY5Y168 Hours After Treatment with Geldanamycin (0.1 nM) and/or FK506 (1 nMor 10 nM) in the Presence of NGF Treatment Mean Length (μM) S.E.M.Untreated 31.86 1.56 NGF (10 ng/mL) 70.38 6.61 Geldanamycin (0.1 nM) +98.07 5.72 NGF (10 ng/mL) FK506 (1 nM) + 89.92 6.40 NGF (10 ng/mL) FK506(10 nM) + 82.68 5.22 NGF (10 ng/mL) Geldanamycin (0.1 nM) + 110.51 6.13FK506 (1 nM) + NGF (10 ng/mL) Geldanamycin (0.1 nM) + 92.50 6.40 FK506(10 nM) + NGF (10 ng/mL)

[0161]

1 2 1 8 PRT Sylvilagus sp. 1 Glu Asp Leu Thr Asp Asp Glu Asp 1 5 2 8 PRTRattus sp. 2 Arg Lys Thr Lys Lys Lys Ile Lys 1 5

What is claimed is:
 1. A pharmaceutical composition comprising a nervegrowth stimulating amount of a non-FKBP12-binding agent that binds to apolypeptide component of a steroid receptor complex other than a steroidhormone binding portion of the complex and a pharmaceutically acceptableexcipient.
 2. The composition of claim 1 wherein binding of the agent tothe polypeptide component causes hsp90 dissociation from the complex orprevents hsp90 association with the complex.
 3. The composition of claim1 wherein the agent is selected from the group consisting of anon-FKBP12-binding FK506 analog, a benzoquinone ansamycin, a peptidecomprising a sequence of a selected polypeptide component of the complexat a site of interaction between the selected component and anotherpolypeptide component of the complex, an antibody that binds to apolypeptide component of the complex, and combinations thereof.
 4. Thecomposition of claim 3 wherein the benzoquinone ansamycin isgeldanamycin or a derivative thereof.
 5. The composition of claim 1wherein the agent binds to hsp90 or FKBP52.
 6. The composition of claim1 wherein the composition further comprises a neurotrophic factor otherthan the agent.
 7. The composition of claim 6 wherein the neurotrophicfactor is selected from the group consisting of NGF, IGF-1, aFGF, bFGF,PDGF, BDNF, CNTF, GDNF, NT-3, NT 4/5, and mixtures thereof.
 8. Thecomposition of claim 1 wherein the composition further comprises asteroid hormone that is a ligand of the steroid receptor complex.
 9. Apharmaceutical composition comprising (i) a nerve growth stimulatingamount of an agent that binds to a polypeptide of a steroid receptorcomplex other than a steroid hormone binding portion of the complex, theagent being selected from the group consisting of a non-FKBP12-bindingFK506 analog, a benzoquinone ansamycin, a peptide comprising a sequenceof a selected polypeptide component of the complex at a site ofinteraction between the selected component and another polypeptidecomponent of the complex, an antibody, and combinations thereof, whereinthe agent causes hsp90 dissociation from the complex or prevents hsp90association with the complex, and (ii) a pharmaceutically acceptableexcipient.
 10. The composition of claim 9 wherein the agent is anon-FKBP12-binding analog of FK506 or a benzoquinone ansamycin.
 11. Thecomposition of claim 9 wherein the component is hsp90 or FKBP52.
 12. Apharmaceutical composition comprising a nerve growth stimulating amountof a non-FKBP12-binding FK506 analog that binds to FKBP52 and apharmaceutically suitable excipient.
 13. A pharmaceutical compositioncomprising a nerve growth stimulating amount of an agent that binds tohsp90 and causes hsp90 dissociation from a steroid receptor complex orprevents hsp90 association with the complex and a pharmaceuticallysuitable excipient.
 14. A method of stimulating nerve cell growth in amammal comprising administering to a mammal the pharmaceuticalcomposition according to claim
 1. 15. A method of stimulating nerve cellgrowth in a mammal comprising administering to a mammal thepharmaceutical composition of claim
 9. 16. A method of stimulating nervecell growth in a mammal comprising administering to a mammal thepharmaceutical composition of claim
 12. 17. A method of stimulatingnerve cell growth in a mammal comprising administering to a mammal thepharmaceutical composition of claim
 13. 18. A method for stimulatinggrowth of a nerve cell comprising contacting the nerve cell with anon-FKBP12-binding agent that binds to a polypeptide component of asteroid receptor complex other than a steroid hormone binding portion ofthe complex and causes hsp90 dissociation from the complex or blocksassociation of hsp90 with the complex.
 19. A method of treating a mammalhaving a transected peripheral nerve or injured spinal cord, the methodcomprising: administering to the mammal a pharmaceutical compositioncomprising a nerve growth stimulating amount of a non-FKBP12-bindingagent that binds to a polypeptide component of a steroid receptorcomplex other than a steroid hormone binding portion of the complex anda pharmaceutically acceptable excipient; and grafting to the peripheralnerve or spinal cord an allograft or an artificial nerve graft.
 20. Themethod of claim 19 wherein grafting the allograft or artificial nervegraft to the peripheral nerve or spinal cord of the mammal produces agap between transected ends of the peripheral nerve or spinal cord,wherein the non-FKBP12-binding agent is administered by filling the gapwith a gap-filling composition comprising the non-FKBP12-binding agent.21. A method of identifying a compound that stimulates nerve cell growthcomprising: assaying a plurality of test compounds for binding to apolypeptide component of a steroid receptor complex other than a steroidhormone binding portion of the complex, thereby identifying bindingcompounds; assaying the binding compounds for stimulation of nerve cellgrowth.
 22. The method of claim 19 further comprising assaying thebinding compounds for dissociation of hsp90 from the complex or blockingassociation of hsp90 with the complex.